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STIM1, an essential and conserved component of store-operated Ca2+ channel function.

Roos J, DiGregorio PJ, Yeromin AV, Ohlsen K, Lioudyno M, Zhang S, Safrina O, Kozak JA, Wagner SL, Cahalan MD, Veliçelebi G, Stauderman KA - J. Cell Biol. (2005)

Bottom Line: RNAi-mediated knockdown of Stim in Drosophila S2 cells significantly reduced TG-dependent Ca2+ entry.Similarly, knockdown of the human homologue STIM1 significantly reduced CRAC channel activity in Jurkat T cells.We propose that STIM1, a ubiquitously expressed protein that is conserved from Drosophila to mammalian cells, plays an essential role in SOC influx and may be a common component of SOC and CRAC channels.

View Article: PubMed Central - PubMed

Affiliation: Torrey Pines Therapeutics, Inc., La Jolla, CA 92037, USA.

ABSTRACT
Store-operated Ca2+ (SOC) channels regulate many cellular processes, but the underlying molecular components are not well defined. Using an RNA interference (RNAi)-based screen to identify genes that alter thapsigargin (TG)-dependent Ca2+ entry, we discovered a required and conserved role of Stim in SOC influx. RNAi-mediated knockdown of Stim in Drosophila S2 cells significantly reduced TG-dependent Ca2+ entry. Patch-clamp recording revealed nearly complete suppression of the Drosophila Ca2+ release-activated Ca2+ (CRAC) current that has biophysical characteristics similar to CRAC current in human T cells. Similarly, knockdown of the human homologue STIM1 significantly reduced CRAC channel activity in Jurkat T cells. RNAi-mediated knockdown of STIM1 inhibited TG- or agonist-dependent Ca2+ entry in HEK293 or SH-SY5Y cells. Conversely, overexpression of STIM1 in HEK293 cells modestly enhanced TG-induced Ca2+ entry. We propose that STIM1, a ubiquitously expressed protein that is conserved from Drosophila to mammalian cells, plays an essential role in SOC influx and may be a common component of SOC and CRAC channels.

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Suppression of STIM1 in HEK293 cells inhibits SOC influx. (A) RT-PCR analysis. STIM1 and STIM2 mRNA levels were reduced in cells transfected with the appropriate siRNA to <50% of control cells (transfected with scrambled siRNA). GAPDH levels were unchanged in either treatment group. (B) Western blot analysis. In cells transfected with the STIM1 siRNA, STIM1 protein levels were reduced to <10% of control levels, whereas GAPDH levels were unchanged. (C) Immunofluorescence localization of STIM1 in HEK293 cells. Nuclear staining pattern (left) with DAPI (Molecular Probes) in HEK293 cells treated with either a scrambled siRNA (top) or siRNA to STIM1 (bottom). No change in nuclear staining pattern or intensity was observed after RNAi-induced suppression of STIM1. STIM1-associated immunofluorescence (right) in HEK293 cells treated with either control (top) or STIM1 (bottom) siRNAs. In control cells, STIM1 has a diffuse reticulated localization pattern with some punctuate staining, which is consistent with expression associated with plasma membrane and ER. The intensity of STIM1 immunofluorescence was markedly decreased in the cells treated with STIM1 siRNA. (D) Calcium signals in HEK293 cells after RNAi-mediated knockdown. Suppression of STIM1 (dotted line) reduced SOC influx by 60% compared with control (solid line; P < 10−4, unpaired t test), whereas suppression of STIM2 (dashed line) had little effect. Data indicate RFUs in 384-well plates monitored in a FLIPR384 fluorimeter. The traces are from a representative experiment, and are averaged signals from 48 wells per group. Traces from cells treated with vehicle (DMSO) instead of TG were essentially flat (not depicted for clarity). (E) Calcium signals after muscarinic receptor activation. RT-PCR analysis revealed that the muscarinic receptor, subtype m3, is expressed in our HEK293 cells (not depicted). 300 μM of methylcholine evoked Ca2+-release transients in Ca2+-free buffer were not inhibited by STIM1 suppression, but SOC influx upon readdition of 2 mM Ca2+ was greatly reduced in STIM1 siRNA-treated cells (dotted line) compared with control cells (solid line). The apparent enhancement of the methylcholine-evoked Ca2+ release transient in the STIM1-suppressed cells was not a consistent finding. (F) TG-induced Ba2+ entry. The rate of TG-induced Ba2+ entry in STIM1-suppressed cells (dotted line) was significantly lower than in control cells (solid line) or STIM2-suppressed cells (dashed line; P < 10−4, unpaired t test).
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fig6: Suppression of STIM1 in HEK293 cells inhibits SOC influx. (A) RT-PCR analysis. STIM1 and STIM2 mRNA levels were reduced in cells transfected with the appropriate siRNA to <50% of control cells (transfected with scrambled siRNA). GAPDH levels were unchanged in either treatment group. (B) Western blot analysis. In cells transfected with the STIM1 siRNA, STIM1 protein levels were reduced to <10% of control levels, whereas GAPDH levels were unchanged. (C) Immunofluorescence localization of STIM1 in HEK293 cells. Nuclear staining pattern (left) with DAPI (Molecular Probes) in HEK293 cells treated with either a scrambled siRNA (top) or siRNA to STIM1 (bottom). No change in nuclear staining pattern or intensity was observed after RNAi-induced suppression of STIM1. STIM1-associated immunofluorescence (right) in HEK293 cells treated with either control (top) or STIM1 (bottom) siRNAs. In control cells, STIM1 has a diffuse reticulated localization pattern with some punctuate staining, which is consistent with expression associated with plasma membrane and ER. The intensity of STIM1 immunofluorescence was markedly decreased in the cells treated with STIM1 siRNA. (D) Calcium signals in HEK293 cells after RNAi-mediated knockdown. Suppression of STIM1 (dotted line) reduced SOC influx by 60% compared with control (solid line; P < 10−4, unpaired t test), whereas suppression of STIM2 (dashed line) had little effect. Data indicate RFUs in 384-well plates monitored in a FLIPR384 fluorimeter. The traces are from a representative experiment, and are averaged signals from 48 wells per group. Traces from cells treated with vehicle (DMSO) instead of TG were essentially flat (not depicted for clarity). (E) Calcium signals after muscarinic receptor activation. RT-PCR analysis revealed that the muscarinic receptor, subtype m3, is expressed in our HEK293 cells (not depicted). 300 μM of methylcholine evoked Ca2+-release transients in Ca2+-free buffer were not inhibited by STIM1 suppression, but SOC influx upon readdition of 2 mM Ca2+ was greatly reduced in STIM1 siRNA-treated cells (dotted line) compared with control cells (solid line). The apparent enhancement of the methylcholine-evoked Ca2+ release transient in the STIM1-suppressed cells was not a consistent finding. (F) TG-induced Ba2+ entry. The rate of TG-induced Ba2+ entry in STIM1-suppressed cells (dotted line) was significantly lower than in control cells (solid line) or STIM2-suppressed cells (dashed line; P < 10−4, unpaired t test).

Mentions: To further examine the role of STIM1 in SOCE in mammalian cells, we generated siRNAs to STIM1 and STIM2 and tested them individually in HEK293 cells on TG-induced Ca2+ influx, previously linked to TRPC1 and TRPC3 proteins (Wu et al., 2000). RT-PCR and Western blot analysis indicated that STIM1 mRNA and protein levels were selectively reduced by the STIM1 siRNA (Fig. 6, A and B). Immunofluorescence localization indicated an expression pattern consistent with plasma membrane and ER localization (Manji et al., 2000) and confirmed the reduction of STIM1 protein by RNAi (Fig. 6 C). Cellular metabolism of the mitochondrial substrate alamarBlue was not altered (unpublished data), indicating the absence of cytotoxicity or mitochondrial stress after STIM1 suppression. In STIM1-suppressed cells, TG-dependent Ca2+ influx was inhibited by 60%, whereas the Ca2+ release transient was unaffected compared with control cells transfected with a nonsilencing scrambled siRNA (Fig. 6 D). In contrast, Ca2+ influx was unaltered in cells treated with the siRNA for STIM2, even though STIM2 mRNA was effectively reduced. In addition to inhibiting TG-evoked Ca2+ influx, knockdown of STIM1 potently inhibited muscarinic receptor-induced Ca2+ influx, but did not reduce IP3-induced Ca2+ release (Fig. 6 E). The store-operated cation entry pathway in HEK293 cells is known to be permeable to Ba2+ (Wu et al., 2000; Trebak et al., 2002). Measurement of Ba2+ influx has the advantage of being a more direct reflection of cation entry via the plasma membrane because it avoids possible effects on cellular buffering of Ca2+ or other Ca2+ regulatory mechanisms. The initial rate of TG-induced Ba2+ entry in cells transfected with STIM1 siRNA was only 17% of control, whereas Ba2+ entry was unaffected in cells transfected with siRNA for STIM2 (Fig. 6 F). In contrast, stable overexpression of STIM1 in HEK293 cells modestly enhanced TG-induced Ca2+ entry by an average of 17% (Fig. 7 B). Interestingly, the increase in SOC influx was small in comparison to the robust increase in STIM1 protein levels (Fig. 7 A), and definitive SOC current was still not detected, nor was there any change in Mg2+-inhibited cation current (Fig. 7, B–G). Thus, although STIM1 is required for SOC influx in HEK293 cells at the level of Ca2+ (or Ba2+) entry across the plasma membrane, overexpression does not appear to greatly enhance the number of activatable SOC channels.


STIM1, an essential and conserved component of store-operated Ca2+ channel function.

Roos J, DiGregorio PJ, Yeromin AV, Ohlsen K, Lioudyno M, Zhang S, Safrina O, Kozak JA, Wagner SL, Cahalan MD, Veliçelebi G, Stauderman KA - J. Cell Biol. (2005)

Suppression of STIM1 in HEK293 cells inhibits SOC influx. (A) RT-PCR analysis. STIM1 and STIM2 mRNA levels were reduced in cells transfected with the appropriate siRNA to <50% of control cells (transfected with scrambled siRNA). GAPDH levels were unchanged in either treatment group. (B) Western blot analysis. In cells transfected with the STIM1 siRNA, STIM1 protein levels were reduced to <10% of control levels, whereas GAPDH levels were unchanged. (C) Immunofluorescence localization of STIM1 in HEK293 cells. Nuclear staining pattern (left) with DAPI (Molecular Probes) in HEK293 cells treated with either a scrambled siRNA (top) or siRNA to STIM1 (bottom). No change in nuclear staining pattern or intensity was observed after RNAi-induced suppression of STIM1. STIM1-associated immunofluorescence (right) in HEK293 cells treated with either control (top) or STIM1 (bottom) siRNAs. In control cells, STIM1 has a diffuse reticulated localization pattern with some punctuate staining, which is consistent with expression associated with plasma membrane and ER. The intensity of STIM1 immunofluorescence was markedly decreased in the cells treated with STIM1 siRNA. (D) Calcium signals in HEK293 cells after RNAi-mediated knockdown. Suppression of STIM1 (dotted line) reduced SOC influx by 60% compared with control (solid line; P < 10−4, unpaired t test), whereas suppression of STIM2 (dashed line) had little effect. Data indicate RFUs in 384-well plates monitored in a FLIPR384 fluorimeter. The traces are from a representative experiment, and are averaged signals from 48 wells per group. Traces from cells treated with vehicle (DMSO) instead of TG were essentially flat (not depicted for clarity). (E) Calcium signals after muscarinic receptor activation. RT-PCR analysis revealed that the muscarinic receptor, subtype m3, is expressed in our HEK293 cells (not depicted). 300 μM of methylcholine evoked Ca2+-release transients in Ca2+-free buffer were not inhibited by STIM1 suppression, but SOC influx upon readdition of 2 mM Ca2+ was greatly reduced in STIM1 siRNA-treated cells (dotted line) compared with control cells (solid line). The apparent enhancement of the methylcholine-evoked Ca2+ release transient in the STIM1-suppressed cells was not a consistent finding. (F) TG-induced Ba2+ entry. The rate of TG-induced Ba2+ entry in STIM1-suppressed cells (dotted line) was significantly lower than in control cells (solid line) or STIM2-suppressed cells (dashed line; P < 10−4, unpaired t test).
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fig6: Suppression of STIM1 in HEK293 cells inhibits SOC influx. (A) RT-PCR analysis. STIM1 and STIM2 mRNA levels were reduced in cells transfected with the appropriate siRNA to <50% of control cells (transfected with scrambled siRNA). GAPDH levels were unchanged in either treatment group. (B) Western blot analysis. In cells transfected with the STIM1 siRNA, STIM1 protein levels were reduced to <10% of control levels, whereas GAPDH levels were unchanged. (C) Immunofluorescence localization of STIM1 in HEK293 cells. Nuclear staining pattern (left) with DAPI (Molecular Probes) in HEK293 cells treated with either a scrambled siRNA (top) or siRNA to STIM1 (bottom). No change in nuclear staining pattern or intensity was observed after RNAi-induced suppression of STIM1. STIM1-associated immunofluorescence (right) in HEK293 cells treated with either control (top) or STIM1 (bottom) siRNAs. In control cells, STIM1 has a diffuse reticulated localization pattern with some punctuate staining, which is consistent with expression associated with plasma membrane and ER. The intensity of STIM1 immunofluorescence was markedly decreased in the cells treated with STIM1 siRNA. (D) Calcium signals in HEK293 cells after RNAi-mediated knockdown. Suppression of STIM1 (dotted line) reduced SOC influx by 60% compared with control (solid line; P < 10−4, unpaired t test), whereas suppression of STIM2 (dashed line) had little effect. Data indicate RFUs in 384-well plates monitored in a FLIPR384 fluorimeter. The traces are from a representative experiment, and are averaged signals from 48 wells per group. Traces from cells treated with vehicle (DMSO) instead of TG were essentially flat (not depicted for clarity). (E) Calcium signals after muscarinic receptor activation. RT-PCR analysis revealed that the muscarinic receptor, subtype m3, is expressed in our HEK293 cells (not depicted). 300 μM of methylcholine evoked Ca2+-release transients in Ca2+-free buffer were not inhibited by STIM1 suppression, but SOC influx upon readdition of 2 mM Ca2+ was greatly reduced in STIM1 siRNA-treated cells (dotted line) compared with control cells (solid line). The apparent enhancement of the methylcholine-evoked Ca2+ release transient in the STIM1-suppressed cells was not a consistent finding. (F) TG-induced Ba2+ entry. The rate of TG-induced Ba2+ entry in STIM1-suppressed cells (dotted line) was significantly lower than in control cells (solid line) or STIM2-suppressed cells (dashed line; P < 10−4, unpaired t test).
Mentions: To further examine the role of STIM1 in SOCE in mammalian cells, we generated siRNAs to STIM1 and STIM2 and tested them individually in HEK293 cells on TG-induced Ca2+ influx, previously linked to TRPC1 and TRPC3 proteins (Wu et al., 2000). RT-PCR and Western blot analysis indicated that STIM1 mRNA and protein levels were selectively reduced by the STIM1 siRNA (Fig. 6, A and B). Immunofluorescence localization indicated an expression pattern consistent with plasma membrane and ER localization (Manji et al., 2000) and confirmed the reduction of STIM1 protein by RNAi (Fig. 6 C). Cellular metabolism of the mitochondrial substrate alamarBlue was not altered (unpublished data), indicating the absence of cytotoxicity or mitochondrial stress after STIM1 suppression. In STIM1-suppressed cells, TG-dependent Ca2+ influx was inhibited by 60%, whereas the Ca2+ release transient was unaffected compared with control cells transfected with a nonsilencing scrambled siRNA (Fig. 6 D). In contrast, Ca2+ influx was unaltered in cells treated with the siRNA for STIM2, even though STIM2 mRNA was effectively reduced. In addition to inhibiting TG-evoked Ca2+ influx, knockdown of STIM1 potently inhibited muscarinic receptor-induced Ca2+ influx, but did not reduce IP3-induced Ca2+ release (Fig. 6 E). The store-operated cation entry pathway in HEK293 cells is known to be permeable to Ba2+ (Wu et al., 2000; Trebak et al., 2002). Measurement of Ba2+ influx has the advantage of being a more direct reflection of cation entry via the plasma membrane because it avoids possible effects on cellular buffering of Ca2+ or other Ca2+ regulatory mechanisms. The initial rate of TG-induced Ba2+ entry in cells transfected with STIM1 siRNA was only 17% of control, whereas Ba2+ entry was unaffected in cells transfected with siRNA for STIM2 (Fig. 6 F). In contrast, stable overexpression of STIM1 in HEK293 cells modestly enhanced TG-induced Ca2+ entry by an average of 17% (Fig. 7 B). Interestingly, the increase in SOC influx was small in comparison to the robust increase in STIM1 protein levels (Fig. 7 A), and definitive SOC current was still not detected, nor was there any change in Mg2+-inhibited cation current (Fig. 7, B–G). Thus, although STIM1 is required for SOC influx in HEK293 cells at the level of Ca2+ (or Ba2+) entry across the plasma membrane, overexpression does not appear to greatly enhance the number of activatable SOC channels.

Bottom Line: RNAi-mediated knockdown of Stim in Drosophila S2 cells significantly reduced TG-dependent Ca2+ entry.Similarly, knockdown of the human homologue STIM1 significantly reduced CRAC channel activity in Jurkat T cells.We propose that STIM1, a ubiquitously expressed protein that is conserved from Drosophila to mammalian cells, plays an essential role in SOC influx and may be a common component of SOC and CRAC channels.

View Article: PubMed Central - PubMed

Affiliation: Torrey Pines Therapeutics, Inc., La Jolla, CA 92037, USA.

ABSTRACT
Store-operated Ca2+ (SOC) channels regulate many cellular processes, but the underlying molecular components are not well defined. Using an RNA interference (RNAi)-based screen to identify genes that alter thapsigargin (TG)-dependent Ca2+ entry, we discovered a required and conserved role of Stim in SOC influx. RNAi-mediated knockdown of Stim in Drosophila S2 cells significantly reduced TG-dependent Ca2+ entry. Patch-clamp recording revealed nearly complete suppression of the Drosophila Ca2+ release-activated Ca2+ (CRAC) current that has biophysical characteristics similar to CRAC current in human T cells. Similarly, knockdown of the human homologue STIM1 significantly reduced CRAC channel activity in Jurkat T cells. RNAi-mediated knockdown of STIM1 inhibited TG- or agonist-dependent Ca2+ entry in HEK293 or SH-SY5Y cells. Conversely, overexpression of STIM1 in HEK293 cells modestly enhanced TG-induced Ca2+ entry. We propose that STIM1, a ubiquitously expressed protein that is conserved from Drosophila to mammalian cells, plays an essential role in SOC influx and may be a common component of SOC and CRAC channels.

Show MeSH
Related in: MedlinePlus